Alfalfa plant and seed corresponding to transgenic event kk 179-2 and methods for detection thereof

ABSTRACT

The present invention provides a transgenic alfalfa event KK 179-2. The invention also provides cells, plant parts, seeds, plants, commodity products related to the event, and DNA molecules that are unique to the event and were created by the insertion of transgenic DNA into the genome of a alfalfa plant. The invention further provides methods for detecting the presence of said alfalfa event nucleotide sequences in a sample, probes and primers for use in detecting nucleotide sequences that are diagnostic for the presence of said alfalfa event.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is entitled to priority pursuant to 35 U.S.C. §119 (e)to U.S. provisional patent application No. 61/503,373, which was filedon Jun. 30, 2011, and U.S. provisional patent application No.61/664,359, which was filed on Jun. 26, 2012, the disclosures of whichare incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing file named “57978_seq_listing.txt”, which is 10,564bytes (measured in MS-WINDOWS) which was electronically filed and whichwas created on May, 1 2012 is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to alfalfa transgenic event KK179-2. Theinvention also provides cells, plant parts, seeds, plants, commodityproducts related to the event, and DNA molecules that are unique to theevent and were created by the insertion of transgenic DNA into thegenome of an alfalfa plant. The invention further provides methods fordetecting the presence of said alfalfa event nucleotide sequences in asample, probes and primers for use in detecting nucleotide sequencesthat are diagnostic for the presence of said alfalfa event.

BACKGROUND OF THE INVENTION

Alfalfa (Medicago sativa) is the most cultivated legume worldwide, withthe US being the top alfalfa producer. The methods of biotechnology havebeen applied to alfalfa for improvement of agronomic traits and thequality of the product. One such agronomic trait is lignin content.

Lignin is the second most abundant terrestrial biopolymer and accountsfor 30% of the organic carbon. Lignin is crucial for structuralintegrity of the cell wall and it imparts stiffness and strength to thestem. Lignin content is inversely correlated with forage digestibilityfor diary cattle. A reduction in lignin may be achieved in transgenicplants by the expression of a RNA suppression construct capable ofproviding such decrease while at the same time provide increased alfalfadigestibility. The expression of foreign genes or suppression moleculesin plants is known to be influenced by many factors, such as theregulatory elements used, the chromosomal location of the transgeneinsert, the proximity of any endogenous regulatory elements close to thetransgene insertion site, and environmental factors such as light andtemperature. For example, it has been observed that there may bevariation in the overall level of transgene suppression or in thespatial or temporal pattern of transgene suppression betweensimilarly-produced events. For this reason, it is often necessary toscreen hundreds of independent transformation events in order toultimately identify one event useful for commercial agriculturalpurposes. Such an event, once identified as having the desiredsuppression phenotype, molecular characteristics and the improved trait,may then be used for introgressing the improved trait into other geneticbackgrounds using plant breeding methods. The resulting progeny wouldcontain the transgenic event and would therefore have the samecharacteristics for that trait of the original transformant. This may beused to produce a number of different crop varieties that comprise theimproved trait and are suitably adapted to specific local growingconditions.

It would be advantageous to be able to detect the presence oftransgene/genomic DNA of a particular plant in order to determinewhether progeny of a sexual cross contain the transgene/genomic DNA ofinterest. In addition, a method for detecting a particular plant wouldbe helpful when complying with regulations requiring the pre-marketapproval and labeling of foods derived from the transgenic crop plants.

The presence or absence of a suppression element may be detected by anywell known nucleic acid detection method such as the polymerase chainreaction (PCR) or DNA hybridization using nucleic acid probes. Thesedetection methods generally focus on frequently used genetic elements,such as promoters, terminators, marker genes, etc. As a result, suchmethods may not be useful for discriminating between differenttransformation events, particularly those produced using the same DNAconstruct unless the sequence of chromosomal DNA adjacent to theinserted DNA (“flanking DNA”) is known. An event-specific PCR assay isdiscussed, for example, by Taverniers et al. (J. Agric. Food Chem., 53:3041-3052, 2005) in which an event-specific tracing system fortransgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73was demonstrated. In this study, event-specific primers and probes weredesigned based upon the sequences of the genome/transgene junctions foreach event. Transgenic plant event specific DNA detection methods havealso been described in U.S. Pat. Nos. 7,632,985; 7,566,817; 7,368,241;7,306,909; 7,718,373; 7,189,514, 7,807,357 and 7,820,392.

SUMMARY OF THE INVENTION

The present invention is an alfalfa transgenic event designated eventKK179-2, having representative seed sample deposited with American TypeCulture Collection (ATCC) under the Accession No. PTA-11833.

The invention provides a plant, seed, cell, progeny plant, or plant partcomprising the event derived from a plant, cell, plant part, or seedcomprising event KK179-2. The invention thus includes, but is notlimited to pollen, ovule, flowers, shoots, roots and leaves.

One aspect of the invention provides compositions and methods fordetecting the presence of a DNA transgenic/genomic junction region fromalfalfa event KK179-2 plant or seed. DNA molecules are provided thatcomprise at least one transgene/genomic junction DNA molecule selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and complementsthereof, wherein the junction molecule spans the insertion site. Analfalfa event KK179-2 and seed comprising these DNA molecules is anaspect of this invention.

A novel DNA molecule is provided that is a DNA transgene/genomic regionSEQ ID NO:3 or the complement thereof, from alfalfa event KK179-2. Analfalfa plant and seed comprising SEQ ID NO: 3 in its genome is anaspect of this invention. In another aspect of the invention, a DNAmolecule is provided that is a DNA transgene/genomic resion SEQ ID NO:4or the complement thereof, wherein this DNA molecule is novel in alfalfaevent KK179-2. An alfalfa plant and seed comprising SEQ ID NO:4 in itsgenome is an aspect of this invention.

The invention provides DNA molecules related to event KK179-2. These DNAmolecules may comprise nucleotide sequences representing or derived fromthe junction of the transgene insertion and flanking genomic DNA ofevent KK179-2, and/or a region of the genomic DNA flanking the insertedDNA, and/or a region of the integrated transgenic DNA flanking theinsertion site, and/or a region of the integrated transgenic expressioncassette, and/or a contiguous sequence of any of these regions. Theinvention also provides DNA molecules useful as primers and probesdiagnostic for the event. Plants, cells, plant parts, commodityproducts, progeny, and seeds comprising these molecules are provided.

According to one aspect of the invention, compositions and methods areprovided for detecting the presence of the transgene/genomic insertionregion from a novel alfalfa plant designated KK179-2. DNA sequences areprovided that comprise at least one junction sequence of KK179-2selected from the group consisting of SEQ ID NO: 1 (corresponding topositions 1038 through 1057 of SEQ ID NO: 6, FIG. 1 [F]), and SEQ ID NO:2 (corresponding to positions 3620 through 3639 of SEQ ID NO: 6, FIG. 1[F]), and complements thereof; wherein a junction sequence is anucleotide sequence that spans the point at which heterologous DNAinserted into the genome is linked to the alfalfa cell genomic DNA anddetection of this sequence in a biological sample containing alfalfa DNAis diagnostic for the presence of the alfalfa event KK179-2 DNA in saidsample (FIG. 1). The alfalfa event KK179-2 and alfalfa seed comprisingthese DNA molecules is an aspect of this invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, wherein a first DNA moleculecomprises a polynucleotide having a nucleotide sequence of sufficientlength of consecutive polynucleotide of any portion of the transgeneregion of the DNA molecule of SEQ ID NO: 3 or SEQ ID NO: 5 and a secondDNA molecule of similar length of any portion of a 5′ flanking alfalfagenomic DNA region of SEQ ID NO: 3, where said DNA molecules function asDNA primers when used together in an amplification reaction with atemplate derived from event KK179-2 to produce an amplicon diagnosticfor event KK179-2 DNA in a sample. Any amplicon produced by DNA primershomologous or complementary to any portion of SEQ ID NO: 3 and SEQ IDNO: 5, and any amplicon that comprises SEQ ID NO: 1 is an aspect of theinvention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, wherein a first DNA moleculecomprises a polynucleotide having a nucleotide sequence of sufficientlength of consecutive polynucleotide of any portion of the transgeneregion of the DNA molecule of SEQ ID NO: 4 or SEQ ID NO: 5 and a secondDNA molecule of similar length of any portion of a 3′ flanking alfalfagenomic DNA of SEQ ID NO: 4, where said DNA molecules function as DNAprimers when used together in an amplification reaction with a templatederived from event KK179-2 to produce an amplicon diagnostic for eventKK179-2 DNA in a sample. Any amplicons produced by DNA primershomologous or complementary to any portion of SEQ ID NO: 4 and SEQ IDNO: 5, and any amplicon that comprises SEQ ID NO: 2 is an aspect of theinvention.

The invention provides methods, compositions, and kits useful fordetecting the presence of DNA derived from alfalfa event KK179-2.Certain methods comprise (a) contacting a sample comprising DNA with aprimer set that when used in a nucleic acid amplification reaction withgenomic DNA from alfalfa event KK179-2 produces an amplicon diagnosticfor the event; (b) performing a nucleic acid amplification reactionthereby producing the amplicon; and (c) detecting the amplicon, whereinsaid amplicon comprises SEQ ID NO: 1 and/or SEQ ID NO: 2. The inventionalso provides a method for detection of the event by (a) contacting asample comprising DNA with a probe that when used in a hybridizationreaction with genomic DNA from the event hybridizes to a DNA moleculespecific for the event; (b) subjecting the sample and probe to stringenthybridization conditions; and (c) detecting the hybridization of theprobe to the DNA molecule. Kits comprising the methods and compositionsof the invention useful for detecting the presence of DNA derived fromthe event are also provided.

The invention further provides a method of producing a alfalfa plantcomprising: (a) crossing a KK179-2 alfalfa plant with a second alfalfaplant, thereby producing a seed; (b) growing said seed to produce aplurality of progeny plants; and (c) selecting a progeny plant thatcomprises KK179-2 or a progeny plant with decreased lignin content.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Diagrammatic representation of the transgenic insert in thegenome of alfalfa event KK179-2; [A] corresponds to the relativepositions of SEQ ID NO: 1 forming the junction between SEQ ID NO: 3 andSEQ ID NO: 5; [B] corresponds to the relative positions of SEQ ID NO: 2forming the junction between SEQ ID NO: 4 and SEQ ID NO: 5; [C]corresponds to the relative position of SEQ ID NO: 3, which contains thealfalfa genomic flanking region and a portion of the arbitrarilydesignated 5′ end of the transgenic DNA insert; [D] corresponds to therelative position of SEQ ID NO: 4, which contains the alfalfa genomeflanking region and a portion of the arbitrarily designated 3′ end ofthe transgenic DNA insert; [E] represents SEQ ID NO: 5, which is thesequence of the transgenic DNA insert including the CCOMT suppressioncassette integrated into the genome of event KK179-2; [F] represents SEQID NO: 6, which is the contiguous sequence comprising, as represented inthe figure from left to right, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:4, in which SEQ ID NOs: 1 and SEQ ID NOs: 2 are incorporated as setforth above, as these sequences are present in the genome in eventKK179-2. LB: refers to the left border of T-DNA; RB: refers to the rightborder of T-DNA.

BRIEF DESCRIPTION OF THE SEQUENCES

The sequence listing file named “57978_seq_listing.txt”, which is 10,564bytes (measured in MS-WINDOWS) which was electronically filed and whichwas created on May, 1 2012 is incorporated herein by reference.

SEQ ID NO: 1—A 20 bp nucleotide sequence representing the left borderjunction between the alfalfa genomic DNA and the integrated DNA insert.This sequence corresponds to positions 1038 through 1057 of SEQ ID NO:6, and to positions 1038 through 1047 of SEQ ID NO: 3 ([C] of FIG. 1).In addition, SEQ ID NO: 1 corresponds to the integrated left border ofthe expression cassette at positions 1 through 10 of SEQ ID NO: 5 ([E]of FIG. 1).

SEQ ID NO: 2—A 20 bp nucleotide sequence representing the right borderjunction between the integrated DNA insert and the alfalfa genomic DNA.This sequence corresponds to positions 3620 to 3639 of SEQ ID NO: 6, andto positions 91 through 111 of SEQ ID NO: 4 ([D] of FIG. 1). Inaddition, SEQ ID NO: 2 corresponds to positions 2573 through 2582 SEQ IDNO: 5 ([E] of FIG. 1).

SEQ ID NO: 3—A 1147 bp nucleotide sequence including the 5′ alfalfagenomic sequence (1047 bp) flanking the inserted DNA of event KK179-2plus a region (100 bp) of the integrated DNA. This sequence correspondsto positions 1 through 1047 of SEQ ID NO: 6.

SEQ ID NO: 4—A 1356 bp nucleotide sequence including the 3′ alfalfagenomic sequence (1256 bp) flanking the inserted DNA of event KK179-2plus a region (100 bp) of the integrated DNA. This sequence correspondsto positions 3529 through 4885 of SEQ ID NO: 6.

SEQ ID NO: 5—The sequence of the integrated expression cassette,including the left and the right border sequences after integration. SEQID NO: 5 corresponds to nucleotide positions 1048 through 3629 of SEQ IDNO: 6.

SEQ ID NO: 6—A 4885 bp nucleotide sequence representing the contig ofthe 5′ sequence flanking the inserted DNA of KK179-2 (SEQ ID NO: 3), thesequence of the integrated DNA insert (SEQ ID NO: 5) and the 3′ sequenceflanking the inserted DNA of KK179-2 (SEQ ID NO: 4).

SEQ ID NO: 7—The sequence of primer SQ20901 used to identify KK179-2event. Production of a 81 bp PCR amplicon using the combination ofprimers SQ20901 and SQ23728 (SEQ ID NO: 8) is a positive result for thepresence of event KK179-2.

SEQ ID NO 8—The sequence of primer SQ223728 used to identify KK179-2event.

SEQ ID NO: 9—The sequence of probe PB10164 used to identify KK179-2event. It is a 6FAM™-labeled synthetic oligonucleotide.

SEQ ID NO: 10—The sequence of primer SQ1532 used as an internal controlin end-point TAQMAN® assays.

SEQ ID NO: 11—The sequence of primer SQ1533 used as an internal controlin end-point TAQMAN® assays.

SEQ ID NO: 12—The sequence of a VIC™-labeled synthetic oligonucleotideprobe PB0359 used as an internal control in end-point TAQMAN® assays.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994.

The present invention provides transgenic alfalfa event KK179-2. Theterm “event” as used herein refers to the plants, seeds, progeny, cells,plant parts thereof, and DNA molecules produced as a result oftransgenic DNA integration into a plant's genome at a particularlocation on a chromosome. Event KK179-2 refers to the plants, seeds,progeny, cells, plant parts thereof, and DNA molecules produced as aresult of the insertion of transgenic DNA having a sequence providedherein as SEQ ID NO: 5 into a particular chromosomal location in theMedicago sativa genome. A seed sample containing KK179-2 has beendeposited with American Type Culture Collection (ATCC) under AccessionNo. PTA-11833.

As used herein, the term “alfalfa” means Medicago sativa and includesall plant varieties that can be bred with alfalfa, including wildalfalfa species. Alfalfa is also called medic, the name of any plant ofthe genus Medicago Old World herbs with blue or yellow flowers similarto those of the related clovers. Unlike corn or soybean, alfalfa plantsare autotetraploid; thus, each trait is determined by genes residing onfour chromosomes instead of two. This complicates genetic research andalso adds to the difficulty of improving alfalfa. Commercial alfalfaseed is often comprised of a mixture of clones that may constitute asynthetic cultivar generated by random interpollination among theselected clones, followed by one to three generations ofopen-pollination in isolation. Additionally, a composite cultivar ofalfalfa may also be developed by blending see of two or more clones orinterpollinating clones in isolation. When forming a composite cultivar,equal quantities of seed from each component clone would be blended toform the initial breeder seed stock.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, such as, a nucleic acid construct that comprises theRNA suppression of a gene of interest, regeneration of a population ofindependently transformed transgenic plants resulting from the insertionof the transgene cassette into the genome of the plant, and selection ofa particular plant with desirable molecular characteristics, such asinsertion of the transgene into a particular genome location. A plantcomprising the event can refer to the original transformant thatincludes the transgene inserted into the particular location in theplant's genome. A plant comprising the event can also refer to progenyof the original transformant that retain the transgene at the sameparticular location in the plant's genome. Such progeny may be producedby a sexual outcross between the transformant, or its progeny, andanother plant. Such another plant may be a transgenic plant comprisingthe same or a different transgene; or may be a non-transgenic plant,such as one from a different variety. Even after repeated back-crossingto a recurrent parent, the event DNA from the transformed parent ispresent in the progeny of the cross at the same genomic location.

A DNA molecule comprising event KK179-2 refers to a DNA moleculecomprising at least a portion of the inserted transgenic DNA (providedas SEQ ID NO: 5) and at least a portion of the flanking genomic DNAimmediately adjacent to the inserted DNA. As such, a DNA moleculecomprising event KK179-2 has a nucleotide sequence representing at leasta portion of the transgenic DNA insert and at least a portion of theparticular region of the genome of the plant into which the transgenicDNA was inserted. The arrangement of the inserted DNA in event KK179-2in relation to the surrounding plant genome is specific and unique toevent KK179-2 and as such the nucleotide sequence of such a DNA moleculeis diagnostic and identifying for event KK179-2. Examples of thesequence of such a DNA molecule are provided herein as SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 6. Such a DNAmolecule is also an integral part of the chromosome of a plant thatcomprises event KK179-2 and may be passed on to progenies of the plant.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether and is the result of human intervention, for example, a DNAmolecule that is comprised of a combination of at least two DNAmolecules heterologous to each other, and/or a DNA molecule that isartificially synthesized and comprises a polynucleotide sequence thatdeviates from the polynucleotide sequence that would normally exist innature, and/or a DNA molecule that comprises a transgene artificiallyincorporated into a host cell's genomic DNA and the associated flankingDNA of the host cell's genome. An example of a recombinant DNA moleculeis a DNA molecule described herein resulting from the insertion of thetransgene into the Medicago sativa genome, which may ultimately resultin the suppression of a recombinant RNA and/or protein molecule in thatorganism. The nucleotide sequence or any fragment derived therefromwould also be considered a recombinant DNA molecule if the DNA moleculecan be extracted from cells, or tissues, or homogenate from a plant orseed or plant tissue; or can be produced as an amplicon from extractedDNA or RNA from cells, or tissues, or homogenate from a plant or seed orplant tissue, any of which is derived from such materials derived fromthe event KK179-2. For that matter, the junction sequences as set forthat SEQ ID NO: 1 and SEQ ID NO: 2, and nucleotide sequences derived fromevent KK179-2 that also contain these junction sequences are consideredto be recombinant DNA, whether these sequences are present within thegenome of the cells of event KK179-2 or present in detectable amounts intissues, progeny, biological samples or commodity products derived fromthe event KK179-2. As used herein, the term “transgene” refers to apolynucleotide molecule artificially incorporated into a host cell'sgenome. Such transgene may be heterologous to the host cell. The term“transgenic plant” refers to a plant comprising such a transgene. A“transgenic plant” includes a plant, plant part, a plant cell or seedwhose genome has been altered by the stable integration of recombinantDNA. A transgenic plant includes a plant regenerated from anoriginally-transformed plant cell and progeny transgenic plants fromlater generations or crosses of a transformed plant. As a result of suchgenomic alteration, the transgenic plant is distinctly different fromthe related wild type plant. An example of a transgenic plant is a plantdescribed herein as comprising event KK179-2.

As used herein, the term “heterologous” refers to a sequence which isnot normally present in a given host genome in the genetic context inwhich the sequence is currently found. In this respect, the sequence maybe native to the host genome, but be rearranged with respect to othergenetic sequences within the host sequence.

The present invention provides DNA molecules and their correspondingnucleotide sequences. As used herein, the terms “DNA sequence”,“nucleotide sequence” and “polynucleotide sequence” refer to thesequence of nucleotides of a DNA molecule, usually presented from the 5′(upstream) end to the 3′ (downstream) end. The nomenclature used hereinis that required by Title 37 of the United States Code of FederalRegulations §1.822 and set forth in the tables in WIPO Standard ST.25(1998), Appendix 2, Tables 1 and 3. The present invention is disclosedwith reference to only one strand of the two nucleotide sequence strandsthat are provided in transgenic event KK179-2. Therefore, by implicationand derivation, the complementary sequences, also referred to in the artas the complete complement or the reverse complementary sequences, arewithin the scope of the present invention and are therefore alsointended to be within the scope of the subject matter claimed.

The nucleotide sequence corresponding to the complete nucleotidesequence of the inserted transgenic DNA and substantial segments of theMedicago sativa genomic DNA flanking either end of the insertedtransgenic DNA is provided herein as SEQ ID NO: 6. A subsection of thisis the inserted transgenic DNA provided as SEQ ID NO: 5. The nucleotidesequence of the genomic DNA flanking the 5′ end of the insertedtransgenic DNA and a portion of the 5′ end of the inserted DNA isprovided herein as SEQ ID NO: 3. The nucleotide sequence of the genomicDNA flanking the 3′ end of the inserted transgenic DNA and a portion ofthe 3′ end of the inserted DNA is provided herein as SEQ ID NO: 4. Theregion spanning the location where the transgenic DNA connects to and islinked to the genomic DNA is referred to herein as the junction. A“junction sequence” or “junction region” refers to a DNA sequence and/orcorresponding DNA molecule that spans the inserted transgenic DNA andthe adjacent flanking genomic DNA. Examples of a junction sequence ofevent KK179-2 are provided herein as SEQ ID NO: 1 and SEQ ID NO: 2. Theidentification of one of these junction sequences in a nucleotidemolecule derived from a alfalfa plant or seed is conclusive that the DNAwas obtained from event KK179-2 and is diagnostic for the presence ofDNA from event KK179-2. SEQ ID NO: 1 is a 20 bp nucleotide sequencespanning the junction between the genomic DNA and the 5′ end of theinserted DNA. SEQ ID NO: 2 is a 20 bp nucleotide sequence spanning thejunction between the genomic DNA and the 3′ end of the inserted DNA. Anysegment of DNA derived from transgenic event KK179-2 that includes theconsecutive nucleotides of SEQ ID NO: 1 is within the scope of thepresent invention. Any segment of DNA derived from transgenic eventKK179-2 that includes the consecutive nucleotides of SEQ ID NO: 2 iswithin the scope of the present invention. In addition, anypolynucleotide molecule comprising a sequence complementary to any ofthe sequences described within this paragraph is within the scope of thepresent invention. FIG. 1 is an illustration of the transgenic DNAinsert in the genome of alfalfa event KK179-2, and the relativepositions of SEQ ID NOs: 1-6 arranged 5′ to 3′.

The present invention further provides exemplary DNA molecules that canbe used either as primers or probes for diagnosing the presence of DNAderived from event KK179-2 in a sample. Such primers or probes arespecific for a target nucleic acid sequence and as such are useful forthe identification of event KK179-2 nucleic acid sequence by the methodsof the invention described herein.

A “probe” is an isolated nucleic acid to which is attached a detectablelabel or reporter molecule, for example, a radioactive isotope, ligand,chemiluminescent agent, or enzyme. Such a probe is complementary to astrand of a target nucleic acid, in the case of the present invention,to a strand of genomic DNA from alfalfa event KK179-2 whether from aalfalfa plant or from a sample that comprises DNA from the event. Probesaccording to the present invention include not only deoxyribonucleic orribonucleic acids but also polyamides and other probe materials thatbind specifically to a target DNA sequence and the detection of suchbinding can be used to diagnose/determine/confirm the presence of thattarget DNA sequence in a particular sample.

A “primer” is typically an isolated polynucleotide that is designed foruse in specific annealing or hybridization methods to hybridize to acomplementary target DNA strand to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya polymerase, for example, a DNA polymerase. A pair of primers may beused with template DNA, such as a sample of Medicago sativa genomic DNA,in a thermal or isothermal amplification, such as polymerase chainreaction (PCR), or other nucleic acid amplification methods, to producean amplicon, where the amplicon produced from such reaction would have aDNA sequence corresponding to sequence of the template DNA locatedbetween the two sites where the primers hybridized to the template. Asused herein, an “amplicon” is a piece or fragment of DNA that has beensynthesized using amplification techniques, such as the product of anamplification reaction. In one embodiment of the invention, an amplicondiagnostic for event KK179-2 comprises a sequence not naturally found inthe Medicago sativa genome. Primer pairs, as used in the presentinvention, are intended to refer to use of two primers binding oppositestrands of a double stranded nucleotide segment for the purpose ofamplifying linearly the polynucleotide segment between the positionstargeted for binding by the individual members of the primer pair,typically in a thermal or isothermal amplification reaction or othernucleic acid amplification methods. Exemplary DNA molecules useful asprimers are provided as SEQ ID NOs: 7-9, may be used as a first DNAmolecule and a second DNA molecule that is different from the first DNAmolecule, and both molecules are each of sufficient length ofconsecutive nucleotides of either SEQ ID NO: 4, SEQ ID NO: 5, or SEQ IDNO: 6 or the complements thereof to function as DNA primers so that,when used together in an amplification reaction with template DNAderived from event KK179-2, an amplicon that is specific and unique totransgenic event KK179-2 would be produced. The use of the term“amplicon” specifically excludes primer-dimers that may be formed in theDNA amplification reaction.

Probes and primers according to the present invention may have completesequence identity to the target sequence, although primers and probesdiffering from the target sequence that retain the ability to hybridizepreferentially to target sequences may be designed by conventionalmethods. In order for a nucleic acid molecule to serve as a primer orprobe it needs only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed. Any nucleic acid hybridization oramplification method can be used to identify the presence of transgenicDNA from event KK179-2 in a sample. Probes and primers are generally atleast about 11 nucleotides, at least about 18 nucleotides, at leastabout 24 nucleotides, and at least about 30 nucleotides or more inlength. Such probes and primers hybridize specifically to a targetsequence under high stringency hybridization conditions.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm the disclosed sequences by knownmethods, for example, by re-cloning and sequencing such sequences.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any nucleic acidhybridization or amplification method can be used to identify thepresence of DNA from a transgenic event in a sample. Nucleic acidmolecules or fragments thereof are capable of specifically hybridizingto other nucleic acid molecules under certain circumstances. As usedherein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least“low-stringency”conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under“high-stringency” conditions. Stringency conditions are described bySambrook et al., 1989, and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In oneembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 1, and SEQ ID NO: 2, or complements or fragments thereof underhigh stringency conditions. The hybridization of the probe to the targetDNA molecule can be detected by any number of methods known to thoseskilled in the art. These can include, but are not limited to,fluorescent tags, radioactive tags, antibody based tags, andchemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (forexample, by PCR) using a particular amplification primer pair,“stringent conditions” are conditions that permit the primer pair tohybridize only to the target nucleic acid sequence to which a primerhaving the corresponding wild-type sequence (or its complement) wouldbind and preferably to produce a unique amplification product, theamplicon, in a DNA amplification reaction. Examples of DNA amplificationmethods include PCR, Recombinase Polymerase Amplification (RPA) (see forexample U.S. Pat No. 7,485,428), Strand Displacement Amplification (SDA)(see for example, U.S. Pat. Nos. 5,455,166 and 5,470,723),Transcription-Mediated Amplification (TMA) (see for example, Guatelli etal., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990)), Rolling CircleAmplification (RCA) (see for example, Fire and Xu, Proc. Natl. Acad Sci.USA 92:4641-4645 (1995); Lui, et al., J. Am. Chem. Soc. 118:1587-1594(1996); Lizardi, et al., Nature Genetics 19:225-232 (1998), U.S. Pat.Nos. 5,714,320 and 6,235,502)), Helicase Dependant Amplification (HDA)(see for example Vincent et al., EMBO Reports 5(8): 795-800 (2004); U.S.Pat. No. 7,282,328), and Multiple Displacement Amplification (MDA) (seefor example Dean et. al., Proc. Natl. Acad Sci. USA 99:5261-5266(2002)).

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, the term “isolated” refers to at least partiallyseparating a molecule from other molecules normally associated with itin its native or natural state. In one embodiment, the term “isolated”refers to a DNA molecule that is at least partially separated from thenucleic acids that normally flank the DNA molecule in its native ornatural state. Thus, DNA molecules fused to regulatory or codingsequences with which they are not normally associated, for example asthe result of recombinant techniques, are considered isolated herein.Such molecules are considered isolated even when integrated into thechromosome of a host cell or present in a nucleic acid solution withother DNA molecules.

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the present invention. For example, PCR (polymerase chain reaction)technology can be used to amplify a particular starting DNA moleculeand/or to produce variants of the original molecule. DNA molecules, orfragments thereof, can also be obtained by other techniques such as bydirectly synthesizing the fragment by chemical means, as is commonlypracticed by using an automated oligonucleotide synthesizer.

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying event KK179-2,selecting plant varieties or hybrids comprising event KK179-2, detectingthe presence of DNA derived from event KK179-2 in a sample, andmonitoring samples for the presence and/or absence of event KK179-2 orplants and plant parts comprising event KK179-2.

The present invention provides plants, progeny, seeds, plant cells,plant parts such as pollen, ovule, pod, flower, root or stem tissue, andleaf. These plants, progeny, seeds, plant cells, plant parts, andcommodity products contain a detectable amount of a polynucleotide ofthe present invention, such as a polynucleotide comprising at least oneof the sequences provided as the consecutive nucleotides of SEQ ID NO:1, and the consecutive nucleotides of SEQ ID NO: 2. Plants, progeny,seeds, plant cells, plant parts and commodity products of the presentinvention may also contain one or more additional suppression targets.

The present invention provides plants, progeny, seeds, plant cells, andplant part such as pollen, ovule, pod, flower, root or stem tissue, andleaf derived from a transgenic plant comprising event KK179-2. Arepresentative sample of seed comprising event KK179-2 has beendeposited according to the Budapest Treaty for the purpose of enablingthe present invention. The repository selected for receiving the depositis the American Type Culture Collection (ATCC) having an address at10801 University Boulevard, Manassas, Va. USA, Zip Code 20110. The ATCCrepository has assigned the accession No. PTA-11833 to event KK179-2seed.

The present invention provides a microorganism comprising a DNA moleculehaving a nucleotide sequence selected from the group consisting of theconsecutive nucleotides of SEQ ID NO: 1, the consecutive nucleotides ofSEQ ID NO: 2. An example of such a microorganism is a transgenic plantcell. Microorganisms, such as a plant cell of the present invention, areuseful in many industrial applications, including but not limited to:(i) use as research tool for scientific inquiry or industrial research;(ii) use in culture for producing endogenous or recombinantcarbohydrate, lipid, nucleic acid, enzymes or protein products or smallmolecules that may be used for subsequent scientific research or asindustrial products; and (iii) use with modern plant tissue culturetechniques to produce transgenic plants or plant tissue cultures thatmay then be used for agricultural research or production. The productionand use of microorganisms such as transgenic plant cells utilizes modernmicrobiological techniques and human intervention to produce a man-made,unique microorganism. In this process, recombinant DNA is inserted intoa plant cell's genome to create a transgenic plant cell that is separateand unique from naturally occurring plant cells. This transgenic plantcell can then be cultured much like bacteria and yeast cells usingmodern microbiology techniques and may exist in an undifferentiated,unicellular state. The new plant cell's genetic composition andphenotype is a technical effect created by the integration of theheterologous DNA into the genome of the cell. Another aspect of thepresent invention is a method of using a microorganism of the presentinvention. Methods of using microorganisms of the present invention,such as transgenic plant cells, include (i) methods of producingtransgenic cells by integrating recombinant DNA into genome of the celland then using this cell to derive additional cells possessing the sameheterologous DNA; (ii) methods of culturing cells that containrecombinant DNA using modern microbiology techniques; (iii) methods ofproducing and purifying endogenous or recombinant carbohydrate, lipid,nucleic acid, enzymes or protein products from cultured cells; and (iv)methods of using modern plant tissue culture techniques with transgenicplant cells to produce transgenic plants or transgenic plant tissuecultures.

As used herein, “progeny” includes any plant, seed, plant cell, and/orregenerable plant part comprising the event DNA derived from an ancestorplant and/or a polynucleotide having at least one of the sequencesprovided as the consecutive nucleotides of SEQ ID NO: 1 or theconsecutive nucleotides of SEQ ID NO: 2. Plants, progeny, and seeds mayheterozygous for the presence of the transgenic sequence. Progeny may begrown from seeds produced by a plant comprising event KK179-2 and/orfrom seeds produced by a plant fertilized with pollen from a plantcomprising event KK179-2.

Progeny plants may be outcrossed, for example, bred with another plant,to produce a varietal or a hybrid seed or plant. The other plant may betransgenic or nontransgenic. A varietal or hybrid seed or plant of thepresent invention may thus be derived by crossing a first parent thatlacks the specific and unique DNA of event KK179-2 with a second parentcomprising event KK179-2, resulting in a hybrid comprising the specificand unique DNA of event KK179-2. Each parent can be a hybrid or aninbred/variety, so long as the cross or breeding results in a plant orseed of the present invention, such as, a seed having at least oneallele comprising the specific and unique DNA of event KK179-2 and/orthe consecutive nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, for example, Fehr,in Breeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

Sexually crossing one plant with another plant, such as,cross-pollinating, may be accomplished or facilitated by humanintervention, for example: by human hands collecting the pollen of oneplant and contacting this pollen with the style or stigma of a secondplant; by human hands and/or human actions removing, destroying, orcovering the stamen or anthers of a plant (for example, by manualintervention or by application of a chemical gametocide) so that naturalself-pollination is prevented and cross-pollination would have to takeplace in order for fertilization to occur; by human placement ofpollinating insects in a position for “directed pollination” (forexample, by placing beehives in orchards or fields or by caging plantswith pollinating insects); by human opening or removing of parts of theflower to allow for placement or contact of foreign pollen on the styleor stigma; by selective placement of plants (for example, intentionallyplanting plants in pollinating proximity); and/or by application ofchemicals to precipitate flowering or to foster receptivity (of thestigma for pollen).

In practicing this method, the step of sexually crossing one plant withitself, such as, self-pollinating or selfing, may be accomplished orfacilitated by human intervention, for example: by human handscollecting the pollen of the plant and contacting this pollen with thestyle or stigma of the same plant and then optionally preventing furtherfertilization of the plant; by human hands and/or actions removing,destroying, or covering the stamen or anthers of other nearby plants(for example, by detasseling or by application of a chemical gametocide)so that natural cross-pollination is prevented and self-pollinationwould have to take place in order for fertilization to occur; by humanplacement of pollinating insects in a position for “directedpollination” (for example, by caging a plant alone with pollinatinginsects); by human manipulation of the flower or its parts to allow forself-pollination; by selective placement of plants (for example,intentionally planting plants beyond pollinating proximity); and/or byapplication of chemicals to precipitate flowering or to fosterreceptivity (of the stigma for pollen).

The present invention provides a plant part that is derived from a plantcomprising event KK179-2. As used herein, a “plant part” refers to anypart of a plant that is comprised of material derived from a plantcomprising event KK179-2. Plant parts include but are not limited topollen, ovule, pod, flower, root or stem tissue, fibers, and leaf. Plantparts may be viable, nonviable, regenerable, and/or non-regenerable.

The present invention provides a commodity product that is derived froma plant comprising event KK179-2. As used herein, a “commodity product”refers to any composition or product that is comprised of materialderived from a plant, seed, plant cell, or plant part comprising eventKK179-2. Commodity products may be sold to consumers and may be viableor nonviable. Nonviable commodity products include but are not limitedto nonviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animal consumption, oil, meal, flour, flakes, bran,fiber, and any other food for human consumption; and biomasses and fuelproducts. Processed alfalfas are the largest source of forage legume inthe world. A plant comprising event KK179-2 can thus be used tomanufacture any commodity product typically acquired from an alfalfaplant. Any such commodity product that is derived from the plantscomprising event KK179-2 may contain at least a detectable amount of thespecific and unique DNA corresponding to event KK179-2, and specificallymay contain a detectable amount of a polynucleotide having a nucleotidesequence of the consecutive nucleotides of SEQ ID NO: 1 and theconsecutive nucleotides of SEQ ID NO: 2. Any standard method ofdetection for polynucleotide molecules may be used, including methods ofdetection disclosed herein. A commodity product is within the scope ofthe present invention if there is any detectable amount of theconsecutive nucleotides of SEQ ID NO: 1 or the consecutive nucleotidesof SEQ ID NO: 2, in the commodity product.

The plant, progeny, seed, plant cell, plant part (such as pollen, ovule,pod, flower, root or stem tissue, and leaf), and commodity products ofthe present invention are therefore useful for, among other things,growing plants for the purpose of producing seed and/or plant partscomprising event KK179-2 for agricultural purposes, producing progenycomprising event KK179-2 for plant breeding and research purposes, usewith microbiological techniques for industrial and researchapplications, and sale to consumers.

The present invention provides methods for producing plants with reducedlignin and plants comprising event KK179-2. Event KK179-2 plant wasproduced by an Agrobacterium mediated transformation method similar tothat described in U.S. Pat. No. 5,914,451, using an inbred alfalfa linewith the construct pFG118. Construct pFG118 contains a plant suppressioncassette for downregulation of the CCOMT enzyme in alfalfa plant cells.Transgenic alfalfa cells were regenerated into intact alfalfa plants andindividual plants were selected from the population of independentlytransformed transgenic plants that showed desirable molecularcharacteristics, such as, the integrity of the transgene cassette,absence of the construct backbone sequence, loss of the unlinkedkanamycin resistance selection cassette. Furthermore, inverse PCR andDNA sequence analyses were performed to determine the 5′ and 3′insert-to-plant genome junctions, to confirm the organization of theelements within the insert (FIG. 1), and to determine the complete DNAsequence of the insert in alfalfa event KK179-2 (SEQ ID NO: 5). Inaddition, transgenic plants were screened and selected for reducedlignin under field conditions. An alfalfa plant that contains in itsgenome the suppression cassette of pFG118 is an aspect of the presentinvention.

Methods for producing a plant with reduced lignin comprising transgenicevent KK179-2 are provided. Transgenic plants used in these methods maybe heterozygous for the transgene. Progeny plants produced by thesemethods may be varietal or hybrid plants; may be grown from seedsproduced by a plant and/or from seed comprising event KK179-2 producedby a plant fertilized with pollen from a plant comprising event KK179-2;and may be homozygous or heterozygous for the transgene. Progeny plantsmay be subsequently self-pollinated to generate a true breeding line ofplants, such as, plants homozygous for the transgene, or alternativelymay be outcrossed, for example, bred with another unrelated plant, toproduce a varietal or a hybrid seed or plant. As used herein, the term“zygosity” refers to the similarity of DNA at a specific chromosomallocation (locus) in a plant. In the present invention, the DNAspecifically refers to the transgene insert along with the junctionsequence (event DNA). A plant is homozygous if the transgene insert withthe junction sequence is present at the same location on each chromosomeof a chromosome pair (4 alleles). A plant is considered heterozygous ifthe transgene insert with the junction sequence is present on only onechromosome of a chromosome pair (1 allele). A wild-type plant is nullfor the event DNA.

Progeny plants and seeds encompassed by these methods and produced byusing these methods are distinct from other plants, for example, becausethe progeny plants and seeds are recombinant and as such created byhuman intervention; contain at least one allele that consists of thetransgenic DNA of the present invention; and/or contain a detectableamount of a polynucleotide sequence selected from the group consistingof consecutive nucleotides of SEQ ID NO: 1, or consecutive nucleotidesof SEQ ID NO: 2. A seed may be selected from an individual progenyplant, and so long as the seed comprises SEQ ID NO: 1, or SEQ ID NO: 2,it will be within the scope of the present invention.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, pod, flower, root or stem tissue, and leaves), and commodityproducts of the present invention may be evaluated for DNA composition,gene expression, and/or protein expression. Such evaluation may be doneby using various methods such as PCR, sequencing, northern blotting,southern analysis, western blotting, immuno-precipitation, and ELISA orby using the methods of detection and/or the detection kits providedherein.

Methods of detecting the presence of compositions specific to eventKK179-2 in a sample are provided. One method consists of detecting thepresence of DNA specific to and derived from a cell, a tissue, a seed, aplant or plant parts comprising event KK179-2. The method provides for atemplate DNA sample to be contacted with a primer pair that is capableof producing an amplicon from event KK179-2 DNA upon being subjected toconditions appropriate for amplification, particularly an amplicon thatcomprises SEQ ID NO: 1, and/or SEQ ID NO: 2, or the complements thereof.The amplicon is produced from a template DNA molecule derived from eventKK179-2, so long as the template DNA molecule incorporates the specificand unique nucleotide sequences of SEQ ID NO: 1, or SEQ ID NO: 2. Theamplicon may be single or double stranded DNA or RNA, depending on thepolymerase selected for use in the production of the amplicon. Themethod provides for detecting the amplicon molecule produced in any suchamplification reaction, and confirming within the sequence of theamplicon the presence of the nucleotides corresponding to SEQ ID NO: 1,or SEQ ID NO: 2, or the complements thereof. The detection of thenucleotides corresponding to SEQ ID NO: 1, and/or SEQ ID NO: 2, or thecomplements thereof in the amplicon are determinative and/or diagnosticfor the presence of event KK179-2 specific DNA and thus biologicalmaterial comprising event KK179-2 in the sample.

Another method is provided for detecting the presence of a DNA moleculecorresponding to SEQ ID NO: 3 or SEQ ID NO: 4 in a sample consisting ofmaterial derived from plant or plant tissue. The method consists of (i)obtaining a DNA sample from a plant, or from a group of differentplants, (ii) contacting the DNA sample with a DNA probe moleculecomprising the nucleotides as set forth in either SEQ ID NO: 1 or SEQ IDNO: 2, (iii) allowing the probe and the DNA sample to hybridize understringent hybridization conditions, and then (iv) detecting ahybridization event between the probe and the target DNA sample.Detection of the hybrid composition is diagnostic for the presence ofSEQ ID NO: 3 or SEQ ID NO: 4, as the case may be, in the DNA sample.Absence of hybridization is alternatively diagnostic of the absence ofthe transgenic event in the sample if the appropriate positive controlsare run concurrently. Alternatively, determining that a particular plantcontains either or both of the sequences corresponding to SEQ ID NO: 1or SEQ ID NO: 2, or the complements thereof, is determinative that theplant contains at least one allele corresponding to event KK179-2.

It is thus possible to detect the presence of a nucleic acid molecule ofthe present invention by any well known nucleic acid amplification anddetection methods such as polymerase chain reaction (PCR), recombinasepolymerase amplification (RPA), or DNA hybridization using nucleic acidprobes. An event-specific PCR assay is discussed, for example, byTaverniers et al. (J. Agric. Food Chem., 53: 3041-3052, 2005) in whichan event-specific tracing system for transgenic maize lines Bt11, Bt176,and GA21 and for transgenic event RT73 is demonstrated. In this study,event-specific primers and probes were designed based upon the sequencesof the genome/transgene junctions for each event. Transgenic plant eventspecific DNA detection methods have also been described in U.S. Pat.Nos. 7,632,985; 7,566,817; 7,368,241; 7,306,909; 7,718,373; 7,189,514,7,807,357 and 7,820,392.

DNA detection kits are provided. One type of kit contains at least oneDNA molecule of sufficient length of contiguous nucleotides of SEQ IDNO: 3, SEQ ID NO: 5, or SEQ ID NO: 6 to function as a DNA primer orprobe specific for detecting the presence of DNA derived from transgenicevent KK179-2 in a sample. The DNA molecule being detected with the kitcomprises contiguous nucleotides of the sequence as set forth in SEQ IDNO: 1. Alternatively, the kit may contain at least one DNA molecule ofsufficient length of contiguous nucleotides of SEQ ID NO: 4, SEQ ID NO:5, or SEQ ID NO: 6 to function as a DNA primer or probe specific fordetecting the presence of DNA derived from transgenic event KK179-2 in asample. The DNA molecule being detected with the kit comprisescontiguous nucleotides as set forth in SEQ ID NO: 2.

An alternative kit employs a method in which the target DNA sample iscontacted with a primer pair as described above, then performing anucleic acid amplification reaction sufficient to produce an ampliconcomprising the consecutive nucleotides of SEQ ID NO: 1, and SEQ ID NO:2. Detection of the amplicon and determining the presence of theconsecutive nucleotides of SEQ ID NO: 1, and SEQ ID NO: 2 or thecomplements thereof within the sequence of the amplicon is diagnosticfor the presence of event KK179-2 specific DNA in a DNA sample.

A DNA molecule sufficient for use as a DNA probe is provided that isuseful for determining, detecting, or for diagnosing the presence oreven the absence of DNA specific and unique to event KK179-2 DNA in asample. The DNA molecule contains the consecutive nucleotides of SEQ IDNO: 1, or the complement thereof, and the consecutive nucleotides of SEQID NO: 2, or the complement thereof.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification methods known in the art, including thermaland isothermal amplification methods. The sequence of the heterologousDNA insert, junction sequences, or flanking sequences from event KK179-2(with representative seed samples comprising event KK179-2 deposited asATCC PTA-11883) can be verified by amplifying such sequences from theevent using primers derived from the sequences provided herein followedby standard DNA sequencing of the amplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following thermal amplification of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded amplicon canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. Detection of a fluorescent or other signal indicates thepresence of the insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to a single-strandedamplicon from the region of interest (one primer in the insertedsequence and one in the flanking genomic sequence) and incubated in thepresence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. ddNTPs are added individuallyand the incorporation results in a light signal which is measured. Alight signal indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single ormulti-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon.Using this method an oligonucleotide is designed which overlaps thegenomic flanking and inserted DNA junction. The oligonucleotide ishybridized to single-stranded amplicon from the region of interest (oneprimer in the inserted DNA and one in the flanking genomic DNA sequence)and incubated in the presence of a DNA polymerase and afluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

TAQMAN® (PE Applied Biosystems, Foster City, Calif.) may also be used todetect and/or quantifying the presence of a DNA sequence using theinstructions provided by the manufacturer. Briefly, a FREToligonucleotide probe is designed which overlaps the genomic flankingand insert DNA junction. The FRET probe and amplification primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe andamplification primers (one primer in the insert DNA sequence and one inthe flanking genomic sequence) are cycled in the presence of athermostable polymerase and dNTPs. Following successful amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties resulting in the production of afluorescent signal. The fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Other described methods, such as, microfluidics (US Patent PublicationNo. 2006068398, U.S. Pat. No. 6,544,734) provide methods and devices toseparate and amplify DNA samples. Optical dyes are used to detect andmeasure specific DNA molecules (WO/05017181). Nanotube devices(WO/06024023) that comprise an electronic sensor for the detection ofDNA molecules or nanobeads that bind specific DNA molecules and can thenbe detected.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for the identification of event KK179-2 in a sample and canbe applied to methods for breeding plants containing the appropriateevent DNA. The kits may contain DNA primers or probes that are similaror complementary to SEQ ID NO: 1-6, or fragments or complements thereof.

The kits and detection methods of the present invention are thereforeuseful for, among other things, identifying event KK179-2, selectingplant varieties or hybrids comprising event KK179-2, detecting thepresence of DNA derived from event KK179-2 in a sample, and monitoringsamples for the presence and/or absence of event KK179-2 or plants,plant parts or commodity products comprising event KK179-2.

The following examples are included to demonstrate examples of certainembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the invention, and thus can be considered to constituteexamples of preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments that are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

EXAMPLES Example 1 Isolation of Flanking Sequences Using Inverse PCR andIdentification of Flanking Sequences by Sequencing

This example describes isolation of the alfalfa genomic DNA sequencesflanking the transgenic DNA insert using inverse PCR for event KK179-2,and identification of the flanking genomic sequences by sequencing.

Sequences flanking the T-DNA insertion in event KK179-2 were determinedusing inverse PCR as described in Ochman et al., 1990 (PCR Protocols: Aguide to Methods and Applications, Academic Press, Inc.). Plant genomicDNA was isolated from both wild-type R2336 and the transgenic line fromtissue grown under greenhouse conditions. Frozen leaf tissue was groundwith a mortar and a pestle in liquid nitrogen or by mechanical grinding,followed by DNA extraction using methods known in the art. This methodcan be modified by one skilled in the art to extract DNA from anytissue, including, but not limited to seed.

An aliquot of DNA from each sample was digested with restrictionendonucleases selected based on restriction analysis of the transgenicDNA. After self-ligation of the restriction fragments, PCR amplificationwas performed using primers designed from the transgenic sequence thatwould amplify sequences extending away from the 5′ and 3′ ends of thetransgenic DNA. A variety of Taq polymerases and amplification systemsmay be used. Table 2 shows an example of PCR amplification for flankingsequence isolation using Phusion High Fidelity DNA Polymerase (Cat. No.F531S or F531L, New England Biolabs), and Thermalcyclers AppliedBiosystems GeneAmp 9700, ABI 9800 Fast Thermal Cycler and MJ Opticon.

TABLE 1 An example of inverse PCR amplification for flanking sequenceisolation. Volume Component PCR master mix 2.9 μl water (per reaction)0.05 μl  Primer 1 (100 μM) 0.05 μl  Primer 1 (100 μM) 5.0 μl 2X PhusionTaq 2.0 μl ligated DNA  10 μl Total Step Condition DNA amplification in1 98° C. 30 sec a fast thermocycler 2 98° C. 5 sec 3 60° C. 10 sec 4 72°C. 2 min 5 Go to step 2 30 times 6 72° C. 4 min 7 10° C. forever 8 End

PCR products were separated by agarose gel electrophoresis and purifiedusing a QIAGEN gel purification kit (Qiagen, Valencia, Calif.). Thesubsequent products were sequenced directly using standard sequencingprotocols. Using these two methods, the 5′ flanking sequence, whichextends into the left border sequence of the integrated DNA insertincluding the CCOMT suppression cassette, was identified and ispresented as SEQ ID NO: 3 ([C] of FIG. 1). The 3′ flanking sequence,which extends into the right border sequence of the integrated DNAinsert including the CCOMT suppression cassette, was identified and ispresented as SEQ ID NO: 4 ([D] of FIG. 1). The transgenic DNA integratedinto the R2336 genomic DNA is presented as SEQ ID NO: 5 ([E] of FIG. 1).

The isolated sequences were compared to the T-DNA sequence to identifythe flanking sequences and the co-isolated T-DNA fragments. Confirmationof the presence of the expression cassette was achieved by PCR withprimers designed based upon the deduced flanking sequence data and theknown T-DNA sequence. The R2336 wild type sequence corresponding to thesame region in which the T-DNA was integrated in the transformed linewas isolated using primers designed from the flanking sequences inKK179-2. The flanking sequences in KK179-2 and the R2336 wild typesequence were analyzed against multiple nucleotide and proteindatabases. This information was used to examine the relationship of thetransgene to the plant genome and to look at the insertion siteintegrity. The flanking sequence and wild type sequences were used todesign primers for TAQMAN® endpoint assays used to identify the eventsas described in

Example 2 Event-Specific Endpoint TAQMAN®

This example describes an event-specific endpoint TAQMAN® thermalamplification method for identification of event KK179-2 DNA in asample.

Examples of conditions useful with the event KK179-2-specific endpointTAQMAN® method are described in Table 2 and Table 3. The DNA primersused in the endpoint assay are primers SQ20901 (SEQ ID NO: 7) andSQ23728 (SEQ ID NO: 8) and 6-FAM™ labeled oligonucleotide probe PB10164(SEQ ID NO: 9). 6FAM™ is a fluorescent dye product of Applied Biosystems(Foster City, Calif.) attached to the DNA probe. For TAQMAN® MGB (MinorGroove Binding) probes, the 5′exonuclease activity of Taq DNA polymerasecleaves the probe from the 5′-end, between the fluorophore and quencher.When hybridized to the target DNA strand, quencher and fluorophore areseparated enough to produce a fluorescent signal.

Primers SQ20901 (SEQ ID NO: 7) and SQ23728 (SEQ ID NO: 8) when used asdescribed with probe PB10164 (SEQ ID NO: 9) produce an amplicon of 81 ntthat is diagnostic for event KK179-2 DNA. The analysis includes apositive control from alfalfa known to contain event KK179-2 DNA, anegative control from non-transgenic alfalfa and a negative control thatcontains no template DNA.

These assays are optimized for use with Applied Biosystems GeneAmp PCRSystem 9700, ABI 9800 Fast Thermal Cycler and MJ Research DNA EnginePTC-225. Other methods and apparatus known to those skilled in the artmay be used to produce amplicons that identify the event KK179-2 DNA.

TABLE 2 Alfalfa KK179-2 Event-Specific Endpoint TAQMAN ® PCR ConditionsStep Reagent Volume Comments 1 18 megohm water adjust for final volumeof 10 μl 2 2X Universal Master Mix 5.0 μl 1X final (dNTPs, enzyme andbuffer) concentration of dNTPs, enzyme and buffer 3 Event PrimersSQ20901 and 0.5 μl 1.0 μM final SQ23728 Mix (resuspended concentrationin 18 megohm water to a concentration of 20 μM for each primer) Example:In a microcentrifuge tube, the following are added to achieve 500 μl ata final concentration of 20 μM: 100 μl of Primer SQ20901 at aconcentration of 100 μM 100 μl of Primer SQ23728 at a concentration of100 μM 300 μl of 18 megohm water 4 Event 6-FAM ™ MGB Probe 0.2 μl 0.2 μMfinal PB10164 concentration (resuspended in 18 megohm water to aconcentration of 10 μM) 5 Internal control Primer-1 and 0.5 μl 1 μMfinal internal control Primer-2 Mix concentration (resuspended in 18megohm water to a concentration of 20 μM for each primer) 6 Internalcontrol VIC ™ probe 0.2 μl 0.2 μM final (resuspended in 18 megohmconcentration water to a concentration of 10 μM) 7 Extracted DNA(template): 3.0 μl 1. Leaf or seed samples to be analyzed 2. Negativecontrol (non-transgenic DNA) 3. Negative water control (no templatecontrol) 4. Positive control (KK179-2 DNA)

TABLE 3 Endpoint TAQMAN ® thermocycler conditions Cycle No. Settings 150° C. 2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64° C. 1minute (−1° C./cycle) 30 95° C. 15 seconds 54° C. 1 minute 1 10° C.Forever

A deposit of representative alfalfa event KK179-2 seed disclosed aboveand recited in the claims, has been made under the Budapest Treaty withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110. The ATCC accession number is PTA-11833. The depositwill be maintained in the depository for a period of 30 years, or 5years after the last request, or for the effective life of the patent,whichever is longer, and will be replaced as necessary during thatperiod.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

Example 3 ADL Measurements in the Lower Stem of Reduced Lignin AlfalfaEvents

TABLE 4 Lower stem ADL measurements for the 6 reduced lignin alfalfaevents in two fall dormant (FD) germplasms from 3 locations in 2008Delta Delta Dormancy Event LCI @ UCI @ % Event germplasm Mean Delta 90%90% Diff. P-value JJ041 FD 7.91 −1.75 −1.97 −1.52 −18.09 <.001 JJ266 FD7.48 −2.18 −2.39 −1.98 −22.60 <.001 KK136 FD 7.01 −2.64 −2.90 −2.39−27.40 <.001 KK179 FD 7.65 −2.01 −2.24 −1.79 −20.83 <.001 KK376 FD 7.37−2.29 −2.55 −2.04 −23.75 <.001 KK465 FD 7.30 −2.36 −2.59 −2.13 −24.44<.001 JJ041 FD 7.71 −1.77 −2.01 −1.53 −18.70 <.001 JJ266 FD 6.98 −2.50−2.74 −2.26 −26.38 <.001 KK136 FD 7.38 −2.10 −2.34 −1.86 −22.14 <.001KK179 FD 7.56 −1.92 −2.16 −1.68 −20.24 <.001 KK376 FD 6.51 −2.97 −3.21−2.73 −31.33 <.001 KK465 FD 7.33 −2.15 −2.39 −1.91 −22.68 <.001Abbreviations used in the tables that follow: ADL = Acid DetergentLignin, % of dry matter LSD = Least Significant Difference FD = FallDormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta =difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

Event positive plants in Table 4 showed a significant (p≦0.05) decreasein lower stem ADL which ranged from 18-31% when compared to the poolednegative control. KK179-2 alfalfa event has the reduced lignin phenotypeidentified by the “sweet spot” selection method.

TABLE 5 Lower stem ADL measurements for the 6 reduced lignin alfalfalead events in fall dormant (FD) germplasms grown in 4 locations in2009. Delta Delta Event Control LCI @ UCI @ Event Mean Mean Delta 90%90% % Diff. P-value JJ041 9.46 10.79 −1.32 −1.55 −1.09 −12.26 <.001JJ266 8.53 10.79 −2.26 −2.47 −2.05 −20.95 <.001 KK136 8.52 10.79 −2.27−2.53 −2.02 −21.06 <.001 KK179 8.52 10.79 −1.96 −2.53 −1.74 −18.20 <.001KK376 8.49 10.79 −2.29 −2.54 −2.04 −21.26 <.001 KK465 8.55 10.79 −2.24−2.47 −2.00 −20.73 <.001 Abbreviations used in the tables that follow:ADL = Acid Detergent Lignin, % of dry matter LSD = Least SignificantDifference FD = Fall Dormant KK179 = KK179-2 reduced lignin alfalfa leadevent Delta = difference between Event and Control means (Event −Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

TABLE 6 Lower stem ADL measurements for the 6 reduced lignin alfalfalead events in fall dormant (FD) germplasms grown at 2 locations in 2009Delta Delta Event Control LCI @ UCI @ Event Mean Mean Delta 90% 90% %Diff. P-value JJ041 9.42 11.73 −2.31 −2.61 −2.02 −19.72 <.001 JJ266 8.8411.73 −2.89 −3.18 −2.59 −24.61 <.001 KK136 9.27 11.73 −2.46 −2.79 −2.12−20.94 <.001 KK179 9.45 11.73 −1.28 −2.57 −1.98 −19.41 <.001 KK376 8.7311.73 −3.00 −3.30 −2.70 −25.57 <.001 KK465 9.17 11.73 −2.56 −2.85 −2.27−21.84 <.001 Abbreviations used in the tables that follow: ADL = AcidDetergent Lignin, % of dry matter LSD = Least Significant Difference FD= Fall Dormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta =difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

TABLE 7 Lower stem ADL measurements for the 6 reduced lignin alfalfalead events in non dormant (ND) germplasms grown at 4 locations in 2009Delta Delta Event Control LCI @ UCI @ Event Mean Mean Delta 90% 90% %Diff. P-value JJ041 9.31 10.89 −1.58 −1.81 −1.35 −14.50 <.001 JJ266 8.1110.89 −2.79 −3.01 −2.56 −25.58 <.001 KK136 8.55 10.89 −2.34 −2.57 −2.11−21.50 <.001 KK179 8.87 10.89 −2.03 −2.26 −1.80 −18.61 <.001 KK376 8.2610.89 −2.63 −2.86 −2.40 −24.14 <.001 KK465 9.09 10.89 −1.81 −2.03 −1.58−16.58 <.001 Abbreviations used in the tables that follow: ADL = AcidDetergent Lignin, % of dry matter LSD = Least Significant Difference ND= Non Dormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta =difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

TABLE 8 Lower stem ADL measurements for 6 reduced lignin alfalfa leadevents in non dormant (ND) germplasms grown at 2 locations in 2009.Delta Delta Event Control LCI @ UCI @ Event Mean Mean Delta 90% 90% %Diff. P-value JJ041 8.91 11.16 −2.26 −2.64 −1.87 −20.21 <.001 JJ266 8.5311.16 −2.63 −3.02 −2.25 −23.61 <.001 KK136 8.85 11.16 −2.31 −2.69 −1.92−20.67 <.001 KK179 8.75 11.16 −2.41 −2.80 −2.02 −21.58 <.001 KK376 8.3511.16 −2.81 −3.20 −2.42 −25.16 <.001 KK465 9.14 11.16 −2.03 −2.41 −1.64−18.15 <.001 Abbreviations used in the tables that follow: ADL = AcidDetergent Lignin, % of dry matter LSD = Least Significant Difference ND= Non Dormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta =difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).Tables 6-8 show 2009 data for lower stem ADL in a fall dormant (FD) andnon dormant (ND) germplasms at 4 and 2 locations respectively. The 6event positive lines showed a significant (p≦0.05) reduction in ADLranging from 12-26% when compared to the pooled negative control, withthe lead event KK179 showing a reduction in ADL of 18-22%.

Example 4 NDFD Measurements in the Lower Stem of Reduced Lignin AlfalfaEvents

TABLE 9 Lower stem NDFD measurements for the 6 reduced lignin alfalfalead events in fall dormant (FD) germplasms grown at 3 locations in 2008Dor- Delta Delta mancy LCI UCI germ- Event Control @ @ % P- Event plasmMean Mean Delta 90% 90% Diff. value JJ041 FD 32.68 27.70 4.98 4.23 5.7317.98 <.001 JJ266 FD 33.58 27.70 5.88 5.20 6.56 21.23 <.001 KK136 FD35.46 27.70 7.76 6.91 8.61 28.01 <.001 KK179 FD 33.52 27.70 5.82 5.086.57 21.02 <.001 KK376 FD 34.12 27.70 6.43 5.57 7.28 23.20 <.001 KK465FD 35.33 27.70 7.63 6.88 8.38 27.55 <.001 JJ041 FD 33.27 27.70 5.56 4.696.44 20.08 <.001 JJ266 FD 34.98 27.70 7.27 6.40 8.15 26.25 <.001 KK136FD 34.29 27.70 6.59 5.71 7.46 23.77 <.001 KK179 FD 33.13 27.70 5.42 4.546.30 19.57 <.001 KK376 FD 37.44 27.70 9.74 8.86 10.61  35.14 <.001 KK465FD 35.34 27.70 7.64 6.76 8.52 27.57 <.001 Abbreviations used in thetables that follow: NDFD = Neutral Detergent Fiber Digestibility, % ofNDF (NDF = neutral detergent fiber. Represents the indigestible andslowly digestible components in plant cell wall (cellulose,hemicellulose, lignin (units = % of dry matter)) FD = Fall Dormant KK179= KK179-2 reduced lignin alfalfa lead event Delta = difference betweenEvent and Control means (Event − Control) % Diff = Percent differencebetween Event and Control (Delta/Control * 100) Delta LCI @ 90% = LowerConfidence Interval of Delta value using an alpha level of 0.10 DeltaUCI @ 90% = Upper Confidence Interval of Delta value using an alphalevel of 0.10 P-value = probability of a greater absolute differenceunder the null hypothesis (2-tailed test for significance).

Lower stem NDFD for the 6 reduce lignin events in fall dormant (FD)germplasms at 3 locations. Event positive plants showed a significant(p≦0.05) increase in lower stem NDFD which ranged from 18-35% whencompared to the pooled negative control.

TABLE 10 Lower stem NDFD measurements for the 6 reduced lignin alfalfalead events in fall dormant (FD) germplasms grown at 4 locations in 2009Dor- Delta Delta mancy LCI UCI germ- Event Control @ @ % P- Event plasmMean Mean Delta 90% 90% Diff. value JJ041 FD 28.09 22.31 5.79 4.89 6.6925.95 <.001 JJ266 FD 28.58 22.31 6.27 5.46 7.08 28.11 <.001 KK136 FD28.88 22.31 6.57 5.58 7.56 29.46 <.001 KK179 FD 27.20 22.31 4.90 4.015.78 21.95 <.001 KK376 FD 28.65 22.31 6.34 5.38 7.31 28.43 <.001 KK465FD 28.21 22.31 5.91 4.99 6.83 26.49 <.001 Abbreviations used in thetables that follow: NDFD = Neutral Detergent Fiber Digestibility, % ofNDF (NDF = neutral detergent fiber. Represents the indigestible andslowly digestible components in plant cell wall (cellulose,hemicellulose, lignin (units = % of dry matter)) FD = Fall Dormant KK179= KK179-2 reduced lignin alfalfa lead event Delta = difference betweenEvent and Control means (Event − Control) % Diff = Percent differencebetween Event and Control (Delta/Control * 100) Delta LCI @ 90% = LowerConfidence Interval of Delta value using an alpha level of 0.10 DeltaUCI @ 90% = Upper Confidence Interval of Delta value using an alphalevel of 0.10 P-value = probability of a greater absolute differenceunder the null hypothesis (2-tailed test for significance).

TABLE 11 Lower stem NDFD measurements for the 6 reduced lignin alfalfalead events in non dormant (ND) germplasms grown at 2 locations in 2009Dor- Delta Delta mancy LCI UCI germ- Event Control @ @ % P- Event plasmMean Mean Delta 90% 90% Diff. value JJ041 ND 26.84 20.88 5.96 4.62 7.3028.52 <.001 JJ266 ND 27.79 20.88 6.90 5.63 8.18 33.06 <.001 KK136 ND27.47 20.88 6.59 5.14 8.05 31.56 <.001 KK179 ND 26.73 20.88 5.85 4.517.18 27.99 <.001 KK376 ND 27.19 20.88 6.31 4.97 7.65 30.21 <.001 KK465ND 27.02 20.88 6.14 4.86 7.42 29.41 <.001 Abbreviations used in thetables that follow: NDFD = Neutral Detergent Fiber Digestibility, % ofNDF (NDF = neutral detergent fiber. Represents the indigestible andslowly digestible components in plant cell wall (cellulose,hemicellulose, lignin (units = % of dry matter)) ND = Non Dormant KK179= KK179-2 reduced lignin alfalfa lead event Delta = difference betweenEvent and Control means (Event − Control) % Diff = Percent differencebetween Event and Control (Delta/Control * 100) Delta LCI @ 90% = LowerConfidence Interval of Delta value using an alpha level of 0.10 DeltaUCI @ 90% = Upper Confidence Interval of Delta value using an alphalevel of 0.10 P-value = probability of a greater absolute differenceunder the null hypothesis (2-tailed test for significance).

TABLE 12 Lower stem NDFD measurements for the 6 reduced lignin alfalfalead events in fall dormant (FD) germplasms grown at 4 locations in 2009Dor- Delta Delta mancy LCI UCI germ- Event Control @ @ % P- Event plasmMean Mean Delta 90% 90% Diff. value JJ041 FD 27.96 22.11 5.85 5.01 6.6926.46 <.001 JJ266 FD 29.97 22.11 7.86 7.01 8.70 35.54 <.001 KK136 FD28.84 22.11 6.73 5.89 7.58 30.45 <.001 KK179 FD 27.32 22.11 5.21 4.376.06 23.58 <.001 KK376 ND 29.81 22.11 7.70 6.85 8.54 34.82 <.001 KK465ND 27.37 22.11 5.26 4.41 6.10 23.78 <.001 Abbreviations used in thetables that follow: NDFD = Neutral Detergent Fiber Digestibility, % ofNDF (NDF = neutral detergent fiber. Represents the indigestible andslowly digestible components in plant cell wall (cellulose,hemicellulose, lignin (units = % of dry matter)) FD = Fall Dormant ND =Non Dormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta =difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

TABLE 13 Lower stem NDFD measurements for the 6 reduced lignin alfalfalead events in non dormant (ND) germplasms grown at 2 locations in 2009Dor- Delta Delta mancy LCI UCI germ- Event Control @ @ % P- Event plasmMean Mean Delta 90% 90% Diff. value JJ041 ND 28.10 22.39  5.71 4.15 7.2625.48 <.001 JJ266 ND 28.73 22.39  6.34 4.78 7.89 28.29 <.001 KK136 ND28.66 22.39  6.27 4.71 7.82 28.00 <.001 KK179 ND 27.76 22.39  5.37 3.816.92 23.98 <.001 KK376 ND 29.87 22.39  7.48 5.93 9.04 33.40 <.001 KK465ND 28.95 22.39 56.56 5.00 8.11 29.29 <.001 Abbreviations used in thetables that follow: NDFD = Neutral Detergent Fiber Digestibility, % ofNDF (NDF = neutral detergent fiber. Represents the indigestible andslowly digestible components in plant cell wall (cellulose,hemicellulose, lignin (units = % of dry matter)) ND = Non Dormant KK179= KK179-2 reduced lignin alfalfa lead event Delta = difference betweenEvent and Control means (Event − Control) % Diff = Percent differencebetween Event and Control (Delta/Control * 100) Delta LCI @ 90% = LowerConfidence Interval of Delta value using an alpha level of 0.10 DeltaUCI @ 90% = Upper Confidence Interval of Delta value using an alphalevel of 0.10 P-value = probability of a greater absolute differenceunder the null hypothesis (2-tailed test for significance).Table 11-13 show 2009 data for lower stem NDFD in fall dormant (FD) andnon dormant (ND) germplasm at 4 and 2 locations respectively. The 6event positive reduced lignin alalfa events showed a significant(p≦0.05) increase in NDFD ranging from 22-36% when compared to thepooled negative control, with the lead event KK179-2 showing an increasein NDFD of 22-28%.

Example 5 Vigor Rating for Reduced Lignin Alfalfa Events

TABLE 14 Vigor ratings for the 2 reduced lignin alfalfa events, JJ266and KK179-2 compared to commercial checks and the null controls in 3locations. The reduced lignin event KK179-2 resulted in no off-types forvigor rating scale. Location Location Location Event 1 2 3 Mean JJ2668.0 7.4 7.8 7.7 JJ266, null 7.8 7.4 8.0 7.7 KK179-2 8.0 7.6 7.6 7.7KK179, null 7.4 7.7 8.1 7.7 Commercial Check 1 6.9 6.7 7.1 6.9Commercial Check 2 7.1 7.0 6.7 6.9 Commercial Check 3 7.8 8.1 8.1 8.0Commercial Check 4 7.3 7.6 7.9 7.6

Data collected for these trials are as follows: plant vigor (scored1-10, 10 being best) taken 21 days after previous harvest and the secondweek of May for the spring score, lodging tolerance (scored 1-10, 10being perfectly upright) taken 1-5 days prior to harvest per season.Plant yield (grams of dry matter (DM) per plant) taken after plants weredried, NDFD (using CAI NIR calibration for RL alfalfa) and ADL (usingNIR calibration for RL alfalfa).

Example 6 ADL Measurements in the Whole Plant for Reduced Lignin AlfalfaEvents

TABLE 15 Whole plant hay ADL measurements for the 6 reduced ligninalfalfa lead events in fall dormant (FD) germplasms grown in 4 locationsin 2009 Delta LCI Delta UCI Event Control @ @ % P- Event Mean Mean Delta90% 90% Diff. value JJ041 4.96 5.66 −0.69 −1.55 −0.44 −12.27 <.001 JJ2664.85 5.66 −1.04 −2.47 −0.59 −14.37 <.001 KK136 4.81 5.66 −1.12 −2.53−0.59 −15.09 <.001 KK179 5.11 5.66 −0.80 −2.19 −0.31  −9.79 <.001 KK3764.73 5.66 −1.19 −2.54 −0.66 −16.39 <.001 KK465 5.18 5.66 −0.74 −2.47−0.22  −8.49  0.002 Abbreviations used in the tables that follow: ADL =Acid Detergent Lignin, % of dry matter LSD = Least SignificantDifference FD = Fall Dormant KK179 = KK179-2 reduced lignin alfalfa leadevent Delta = difference between Event and Control means (Event −Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

TABLE 16 Whole plant hay ADL measurements for the 6 reduced ligninalfalfa lead events in non dormant (ND) germplasms grown in 2 locationsin 2009 Delta LCI Delta UCI Event Control @ @ % P- Event Mean Mean Delta90% 90% Diff. value JJ041 5.40 6.16 −0.77 −1.22 −0.31 −12.43 0.006 JJ2665.27 6.16 −0.89 −1.30 −0.48 −14.47 0.000 KK136 5.56 6.16 −0.61 −1.07−0.15  −9.87 0.030 KK179 5.41 6.16 −0.76 −1.19 −0.32 −12.25 0.004 KK3765.20 6.16 −0.97 −1.42 −0.51 −15.66 0.001 KK465 5.57 6.16 −0.60 −1.00−0.19  −9.69 0.016 Abbreviations used in the tables that follow: ADL =Acid Detergent Lignin, % of dry matter LSD = Least SignificantDifference ND = Non Dormant KK179 = KK179-2 reduced lignin alfalfa leadevent Delta = difference between Event and Control means (Event −Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

TABLE 17 Whole plant hay ADL measurements for the 6 reduced ligninalfalfa lead events in fall dormant (FD) germplasms grown in 4 locationsin 2009 Delta LCI Delta UCI Event Control @ @ % P- Event Mean Mean Delta90% 90% Diff. value JJ041 4.93 5.77 −0.85 −1.07 −0.62 −14.64 <0.001JJ266 4.66 5.77 −1.11 −1.33 −0.89 −19.25 <0.001 KK136 5.12 5.77 −0.65−0.88 −0.43 −11.34 <0.001 KK179 5.23 5.77 −0.54 −0.77 −0.32 −9.41 <0.001KK376 4.61 5.77 −1.16 −1.39 −0.93 −20.09 <0.001 KK465 5.28 5.77 −0.49−0.71 −0.26 −8.43 <0.001 Abbreviations used in the tables that follow:ADL = Acid Detergent Lignin, % of dry matter LSD = Least SignificantDifference FD = Fall Dormant KK179 = KK179-2 reduced lignin alfalfa leadevent Delta = difference between Event and Control means (Event −Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed 5 test for significance).

Whole plant ADL data from 2009 across 4 locations is shown in Table 17and 19. The 6 reduced lignin positive events in fall dormant germplasmshowed a significant (p≦0.05) decrease in ADL ranging from 8-19% whencompared to the pooled negative control. Event KK179-2 had a 9.8% and a9.45 reduction in ADL in the fall dormany germplasms respectively.

TABLE 18 Whole plant hay ADL measurements for the 6 reduced ligninalfalfa lead events in non dormant (ND) germplasms grown in 2 locationsin 2009 Delta LCI Delta UCI Event Control @ @ % P- Event Mean Mean Delta90% 90% Diff. value JJ041 5.25 5.94 −0.69 −1.10 −0.28 −11.59  0.006JJ266 4.86 5.94 −1.08 −1.48 −0.69 −18.21 <0.001 KK136 5.57 5.94 −0.37−0.76 −0.02  −6.22  0.123 KK179 5.29 5.94 −0.65 −1.04 −0.25 −10.91 0.007 KK376 5.02 5.94 −0.92 −1.33 −0.51 −15.47 <0.001 KK465 5.37 5.94−0.57 −0.96 −0.18  −9.61  0.018 Abbreviations used in the tables thatfollow: ADL = Acid Detergent Lignin, % of dry matter LSD = LeastSignificant Difference ND = Non Dormant KK179 = KK179-2 reduced ligninalfalfa lead event Delta = difference between Event and Control means(Event − Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

Whole plant ADL data from 2009 across 2 locations is shown in Table 18and 20. The 6 reduced lignin positive events in the non dormantgermplasm showed a significant (p≦0.05) decrease in ADL ranging from10-16% when compared to the pooled negative control. Five of the 6events showed a significant decrease in ADL ranging from 10-18% whencompared to the pooled negative control. Event KK179-2 had 12.3% and10.9% reduction in ADL in the non dormant germplasms respectively.

TABLE 19 Whole plant hay ADL measurements for the reduced lignin alfalfaevent KK179-2 in two fall dormant (FD) germplasms grown in 4 locationsin 2009 compared to commercial checks Dor- Delta Delta Com- mancy LCIUCI mercial Germ- Check @ @ % P- Check plasm KK179 Mean Delta 90% 90%Diff. value 1 FD1 5.22 6.12 −0.90 −1.19 −0.62 −14.77 <.001 2 FD1 5.225.69 −0.47 −0.76 −0.18  −8.31 0.008 3 FD1 5.22 5.38 −0.17 −0.46   0.13 −3.08 0.350 4 FD1 5.22 5.59 −0.38 −0.67 −0.09  −6.75 0.034 1 FD2 5.106.12 −1.02 −1.31 −0.73 −16.67 <.001 2 FD2 5.10 5.69 −0.59 −0.89 −0.29−10.35 0.001 3 FD2 5.10 5.38 −0.28 −0.58   0.02  −5.24 0.119 4 FD2 5.105.59 −0.49 −0.79 −0.20  −8.83 0.006 Abbreviations used in the tablesthat follow: ADL = Acid Detergent Lignin, % of dry matter FD = FallDormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta =difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

TABLE 20 Whole plant hay ADL measurements for the reduced lignin alfalfaevent KK179-2 in two non dormant (ND) germplasm grown in 2 locations in2009 compared to commercial checks Delta Delta Com- LCI UCI mercialGerm- Check @ @ % P- Check plasm KK179-2 Mean Delta 90% 90% Diff. value1 ND1 5.29 5.73 −0.44 −0.96 0.09 −7.62 0.173 2 ND1 5.29 5.81 −0.52 −1.040.01 −8.92 0.106 3 ND1 5.29 5.77 −0.48 −1.01 0.05 −8.34 0.133 4 ND1 5.295.92 −0.63 −1.15 −0.10 −10.61 0.050 1 ND2 5.39 5.73 −0.33 −0.88 0.21−5.77 0.318 2 ND2 5.39 5.81 −0.41 −0.96 0.13 −7.11 0.213 3 ND2 5.39 5.77−0.38 −0.92 0.17 −6.51 0.257 4 ND2 5.39 5.92 −0.52 −1.07 0.02 −8.820.115 Abbreviations used in the tables that follow: ADL = Acid DetergentLignin, % of dry matter ND = Non Dormant KK179 = KK179-2 reduced ligninalfalfa lead event Delta = difference between Event and Control means(Event − Control) % Diff = Percent difference between Event and Control(Delta / Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

Tables 19 and 20 contain whole plant ADL data for the reduced ligninalfalfa event KK179-2 compared to commercial checks. The KK179-2 eventshowed a significant (p≦0.1) decrease in ADL when compared to 3 of the 4fall dormant commercial checks which ranged from 6.8-16.7% (Table 19,data from 4 locations). KK179-2 event in non dormant backgroundgermplasm (ND1) showed a decrease (p≦0.2) in ADL compared to all 4 nondormant commercial checks ranging from 7.6-10.6% (Table 20, data from 2locations). The KK179-2 event in non dormant background germplasm (ND2)showed a overall decrease (p≦0.2) in ADL compared to all 4 non dormantcommercial checks with a significant (p≦0.1) decrease of 8.8% comparedto commercial event 4 (ND2, data from 2 locations).

Example 7 NDFD Measurements in the Whole Plant for Reduced LigninAlfalfa Events

TABLE 21 Whole plant hay NDFD measurements for the 6 reduced ligninalfalfa lead events in fall dormant (FD) germplasms grown in 4 locationsin 2009. Delta Delta Event Control LCI @ UCI @ % P- Event Mean MeanDelta 90% 90% Diff. value JJ041 45.38 39.47 5.90 4.32 7.49 14.96 <0.001JJ266 44.00 39.47 4.53 3.15 5.92 11.48 <0.001 KK136 43.92 39.47 4.452.80 6.10 11.27 <0.001 KK179 42.44 39.47 2.97 1.47 4.47  7.53   0.001KK376 44.82 39.47 5.35 3.71 6.99 13.55 <0.001 KK465 42.13 39.47 2.661.07 4.25  6.74   0.006 Abbreviations used in the tables that follow:NDFD = Neutral Detergent Fiber Digestibility, % of NDF (NDF = neutraldetergent fiber. Represents the indigestible and slowly digestiblecomponents in plant cell wall (cellulose, hemicellulose, lignin (units =% of dry matter)) FD = Fall Dormant KK179 = KK179-2 reduced ligninalfalfa lead event Delta = difference between Event and Control means(Event − Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

TABLE 22 Whole plant hay NDFD measurements for the 6 reduced ligninalfalfa lead events in non dormant (ND) germplasms grown in 2 locationsin 2009 Delta Delta Control LCI @ UCI @ % P- Event Event Mean Mean Delta90% 90% Diff. value JJ041 40.63 35.41 5.23 1.84 8.61 14.76 0.011 JJ26640.81 35.41 5.41 2.35 8.46 15.27 0.004 KK136 38.66 35.41 3.25 −0.19 6.70  9.19 0.120 KK179 40.37 35.41 4.96 1.73 8.19 14.01 0.012 KK37639.75 35.41 4.35 0.96 7.73 12.28 0.035 KK465 38.72 35.41 3.32 0.26 6.37 9.37 0.074 Abbreviations used in the tables that follow: NDFD = NeutralDetergent Fiber Digestibility, % of NDF (NDF = neutral detergent fiber.Represents the indigestible and slowly digestible components in plantcell wall (cellulose, hemicellulose, lignin (units = % of dry matter))ND = Non Dormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta= difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

TABLE 23 Whole plant hay NDFD measurements for the 6 reduced ligninalfalfa lead events in fall dormant (FD) germplasms grown in 4 locationsin 2009. Delta Delta Control LCI @ UCI @ % P- Event Event Mean MeanDelta 90% 90% Diff. value JJ041 44.42 38.96 5.46 3.92 7.00 14.02 <.001JJ266 45.19 38.96 6.22 4.72 7.73 15.98 <.001 KK136 43.63 38.96 4.66 3.166.17 11.97 <.001 KK179 42.56 38.96 3.60 2.10 5.10  9.24 <.001 KK37645.41 38.96 6.45 4.90 7.99 16.54 <.001 KK465 41.52 38.96 2.55 1.05 4.06 6.55   0.005 Abbreviations used in the tables that follow: NDFD =Neutral Detergent Fiber Digestibility, % of NDF (NDF = neutral detergentfiber. Represents the indigestible and slowly digestible components inplant cell wall (cellulose, hemicellulose, lignin (units = % of drymatter)) FD = Fall Dormant KK179 = KK179-2 reduced lignin alfalfa leadevent Delta = difference between Event and Control means (Event −Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

Whole plant NDFD data from 2009 across 4 locations is shown in Table 23and 25. The 6 reduced lignin positive events in fall dormant germplasmshowed a significant (p≦0.05) increase in NDFD ranging from 7-16% whencompared to the pooled negative control. Event KK179-2 had a 7.5% and9.2% increase in NDFD in the fall dormant germplasm respectively.

TABLE 24 Whole plant hay NDFD measurements for the 6 reduced ligninalfalfa lead events in non dormant (ND) germplasms grown in 2 locationsin 2009. Delta Delta Control LCI @ UCI @ % P- Event Event Mean MeanDelta 90% 90% Diff. value JJ041 40.95 37.21 3.74 0.68 6.81 10.06 0.045JJ266 42.06 37.21 4.85 1.92 7.79 13.05 0.007 KK136 40.24 37.21 3.03 0.105.97  8.15 0.089 KK179 41.48 37.21 4.27 1.34 7.21 11.49 0.017 KK37642.22 37.21 5.01 1.95 8.08 13.47 0.007 KK465 40.35 37.21 3.15 0.21 6.08 8.46 0.078 Abbreviations used in the tables that follow: NDFD = NeutralDetergent Fiber Digestibility, % of NDF (NDF = neutral detergent fiber.Represents the indigestible and slowly digestible components in plantcell wall (cellulose, hemicellulose, lignin (units = % of dry matter))ND = Non Dormant KK179 = KK179-2 reduced lignin alfalfa lead event Delta= difference between Event and Control means (Event − Control) % Diff =Percent difference between Event and Control (Delta/Control * 100) DeltaLCI @ 90% = Lower Confidence Interval of Delta value using an alphalevel of 0.10 Delta UCI @ 90% = Upper Confidence Interval of Delta valueusing an alpha level of 0.10 P-value = probability of a greater absolutedifference under the null hypothesis (2-tailed test for significance).

Whole plant NDFD data from 2009 across 2 locations is shown in Table 24and 26. The 6 reduced lignin positive events in non dormant germplasmshowed a significant (p≦0.1) increase in NDFD ranging from 8-15% whencompared to the pooled negative control. Event KK179-2 had a 14.0% and11.5% increase in NDFD in the non dormant germplasm respectively.

TABLE 25 Whole plant hay NDFD measurements for the reduced ligninalfalfa event KK179-2 in two fall dormant (FD) germplasms grown in 4locations in 2009 compared to commercial checks Delta Delta Com- LCI UCImercial Germ- Check @ @ % P- Check plasm KK179 Mean Delta 90% 90% Diff.value 1 FD1 42.17 36.10 6.07 4.27 7.86 16.80  <.001  2 FD1 42.17 40.341.83 0.00 3.66 4.53 0.101 3 FD1 42.17 41.27 0.89 −0.94  2.72 2.16 0.4234 FD1 42.17 38.87 3.29 1.46 5.12 8.47 0.003 1 FD2 42.03 36.10 5.93 4.117.76 16.44  <.001  2 FD2 42.03 40.34 1.70 −0.17  3.56 4.21 0.134 3 FD242.03 41.27 0.76 −1.10  2.63 1.84 0.502 4 FD2 42.03 38.87 3.16 1.30 5.038.13 0.005 Abbreviations used in the tables that follow: NDFD = NeutralDetergent Fiber Digestibility, % of NDF (NDF = neutral detergent fiber.Represents the indigestible and slowly digestible components in plantcell wall (cellulose, hemicellulose, lignin (units = % of dry matter))FD = Fall Dormant KK179 = KK179-2 reduced lignin alfalfa lead eventDelta = difference between Event and Control means (Event − Control) %Diff = Percent difference between Event and Control (Delta/Control *100) Delta LCI @ 90% = Lower Confidence Interval of Delta value using analpha level of 0.10 Delta UCI @ 90% =Upper Confidence Interval of Deltavalue using an alpha level of 0.10 P-value = probability of a greaterabsolute difference under the null hypothesis (2-tailed test forsignificance).

TABLE 26 Whole plant hay NDFD measurements for the reduced ligninalfalfa event KK179-2 in two non dormant (ND) germplasm grown in 2locations in 2009 compared to commercial checks Delta Delta Com- LCI UCImercial Germ- Check @ @ % P- Check plasm KK179 Mean Delta 90% 90% Diff.value 1 ND1 41.46 37.77 3.68 −0.27  7.64 9.75 0.126 2 ND1 41.46 37.124.34 0.39 8.30 11.70 0.071 3 ND1 41.46 34.71 6.74 2.79 10.70 19.43 0.0054 ND1 41.46 35.70 5.75 1.80 9.71 16.12 0.017 1 ND2 40.38 37.77 2.60−1.49  6.70 6.89 0.296 2 ND2 40.38 37.12 3.26 −0.84  7.36 8.79 0.190 3ND2 40.38 34.71 5.66 1.57 9.76 16.31 0.023 4 ND2 40.38 35.70 4.67 0.588.77 13.09 0.061 Abbreviations used in the tables that follow: NDFD =Neutral Detergent Fiber Digestibility, % of NDF (NDF = neutral detergentfiber. Represents the indigestible and slowly digestible components inplant cell wall (cellulose, hemicellulose, lignin (units = % of drymatter)) ND = Non Dormant KK179 = KK179-2 reduced lignin alfalfa leadevent Delta = difference between Event and Control means (Event −Control) % Diff = Percent difference between Event and Control(Delta/Control * 100) Delta LCI @ 90% = Lower Confidence Interval ofDelta value using an alpha level of 0.10 Delta UCI @ 90% = UpperConfidence Interval of Delta value using an alpha level of 0.10 P-value= probability of a greater absolute difference under the null hypothesis(2-tailed test for significance).

Tables 25 and 26 contain whole plant NDFD data for the reduced ligninalfalfa event KK179-2 compared to commercial checks. The KK179-2 eventshowed an increase (p≦0.2) in NDFD when compared to 3 of the 4 falldormant commercial checks which ranged from 4.2-16.8% (Table 25, datafrom 4 locations). KK179-2 event showed an increase (p≦0.2) in NDFDcompared to all 4 non dormant commercial checks (ND1) ranging from9.8-19.4% (Table 26, data from 2 locations). The KK179-2 event showed anincrease (p≦0.2) in NDFD compared to all 4 non dormant commercial checks(ND2), which ranged from 8.8-16.3% (Table 26, data from 2 locations).

Example 8 Yield Across Location Analysis for Reduced Lignin AlfalfaEvents

TABLE 27 Yield across location analysis for 6 reduced lignin events forin fall dormant (FD) and non-dormant (ND) backgrounds compared to poolednegative controls Dor- Delta Delta mancy LCI UCI germ- Event Control @ @% P- Event plasm Year Mean Mean Delta 90% 90% Diff. value JJ041 FD 2008337.56 349.32 −11.76 −42.86 19.35 −3.37 0.532 JJ266 FD 2008 364.72349.32 15.40 −12.94 43.74 4.41 0.370 KK136 FD 2008 306.67 349.32 −42.65−78.12 −7.19 −12.21 0.048 KK179 FD 2008 368.51 349.32 19.19 −11.91 50.305.49 0.309 KK376 FD 2008 354.92 349.32 5.60 −29.85 41.05 1.60 0.794KK465 FD 2008 358.74 349.32 9.42 −21.68 40.53 2.70 0.617 JJ041 FD 20091148.41  1591.58  −143.17 −278.80 −7.55 −9.00 0.083 JJ266 FD 2009 156.501591.58  −26.08 −147.97 95.82 −1.64 0.724 KK136 FD 2009 1468.30 1591.58  −123.28 −269.17 22.61 −7.75 0.164 KK179 FD 2009 1577.84 1591.58  −13.74 −145.29 117.81 −0.86 0.863 KK376 FD 2009 1371.19 1591.58  −220.39 −361.43 −79.35 −13.85 0.011 KK465 FD 2009 1459.44 1591.58  −132.14 −272.37 8.09 −8.30 0.121 JJ041 ND 2009 591.17 764.86−173.70 −292.96 −54.43 −22.71 0.018 JJ266 ND 2009 758.32 764.86 −6.54−119.09 106.01 −0.86 0.923 KK136 ND 2009 771.81 764.86 6.95 −121.20135.10 0.91 0.928 KK179 ND 2009 754.11 764.86 −10.75 −130.04 108.54−1.41 0.881 KK376 ND 2009 584.31 764.86 −180.55 −299.84 −61.25 −23.610.014 KK465 ND 2009 637.67 764.86 −127.20 −239.75 −14.65 −16.63 0.064Abbreviations used in the tables that follow: Yield = Yield calculatedon a per plant basis in grams FD = Fall Dormant ND = Non Dormant KK179 =KK179-2 reduced lignin alfalfa lead event Delta = difference betweenEvent and Control means (Event − Control) % Diff = Percent differencebetween Event and Control (Delta/Control * 100) Delta LCI @ 90% = LowerConfidence Interval of Delta value using an alpha level of 0.10 DeltaUCI @ 90% = Upper Confidence Interval of Delta value using an alphalevel of 0.10 P-value = probability of a greater absolute differenceunder the null hypothesis (2-tailed test for significance).

The data in Table 27 shows the across location yield analysis for the 6reduced lignin events in the fall dormant (FD) and non dormant (ND)germplasms compared to the pooled negative control. There were nosignificant decrease in yield is detected for KK179-2 when compared tothe pooled negative controls.

TABLE 28 Yield across location analysis for Event KK179-2 compared tocommercial checks Dor- Delta Com- macy LCI UCI merical germ- EventControl @ @ % P- Check plasm Year Mean Mean Delta 90% 90% Diff. value 1FD 2008  368.51  239.29 129.22 92.13 166.31 54.00 <.001   2 FD 2008 368.51  308.06  60.45 23.35 97.54 19.62 0.008 3 FD 2008  368.51  349.47 19.05 −18.05 56.14 5.45 0.397 4 FD 2008  368.51  301.09  67.42 30.33104.51 22.39 0.003 1 FD 2009 1361.60 1106.96 254.64 112.59 396.69 23.000.003 2 FD 2009 1361.60 1289.66  71.94 −72.74 216.62 5.58 0.412 3 FD2009 1361.60 1396.58 −34.99  −179.66 109.69 −2.51 0.690 4 FD 20091361.60 1225.01 136.59 −8.09 281.27 11.15 0.120 5 ND 2009  752.63 735.61  17.03 −95.75 129.80 2.31 0.802 6 ND 2009  752.63  803.99 −51.36−164.13 61.42 −6.39 0.451 7 ND 2009  752.63  698.51  54.12 −58.66 166.897.75 0.427 8 ND 2009  752.63  618.75 133.89 21.11 246.66 21.64 0.052Abbreviations used in the tables that follow: Yield = Yield calculatedon a per plant basis in grams FD = Fall Dormant ND = Non Dormant KK179 =KK179-2 reduced lignin alfalfa lead event Delta = difference betweenEvent and Control means (Event − Control) % Diff = Percent differencebetween Event and Control (Delta/Control * 100) Delta LCI @ 90% = LowerConfidence Interval of Delta value using an alpha level of 0.10 DeltaUCI @ 90% = Upper Confidence Interval of Delta value using an alphalevel of 0.10 P-value = probability of a greater absolute differenceunder the null hypothesis (2-tailed test for significance).

Yield data for reduced lignin alfalfa lead event in fall dormant (FD)and non-dormant (ND) germplasms resulted in no significant yielddecrease when compared to 8 commercial checks.

We claim:
 1. A seed of alfalfa plant designated KK179-2 havingrepresentative seed deposited with the American Type Culture Collection(ATCC) with the Patent Deposit Designation PTA-11833.
 2. An alfalfaplant KK179-2 or parts thereof produced by growing the seed of claim 1.3. The alfalfa plant KK179-2 or parts thereof of claim 2, comprisingpollen, ovule, flowers, shoots, roots or leaves.
 4. A progeny plant orparts thereof of the alfalfa plant KK179-2 of claim 2 wherein saidprogeny plant or parts thereof comprise alfalfa event KK179-2.
 5. Theprogeny plant or parts thereof, of claim 4 further comprising pollen,ovule, flower, shoots, roots or leaves.
 6. The alfalfa plant KK179-2 orseed or parts thereof of claim 4, the genome of which produces anamplicon comprising a DNA molecule selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
 7. A recombinantDNA molecule comprising a. a polynucleotide molecule selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 2; b. a polynucleotidemolecule with at least 90% identity to SEQ ID NO: 6; or c. apolynucleotide molecule complementary to a) or b).
 8. The DNA moleculeof claim 7, wherein said DNA molecule is derived from event KK179-2, arepresentative sample of seed having been deposited under ATCC AccessionNO. PTA-11833.
 9. The DNA molecule of claim 7, wherein said DNA moleculeis an amplicon produced from a template molecule derived from DNA fromevent KK179-2.
 10. A polynucleotide probe diagnostic for the presence ofevent KK179-2 DNA, wherein said polynucleotide probe is of sufficientlength to bind to a nucleic acid molecule comprising SEQ ID NO: 1, andSEQ ID NO: 2, or complements thereof, and wherein said polynucleotideprobe hybridizes under stringent hybridization conditions with a DNAmolecule comprising SEQ ID NO: 1, or SEQ ID NO: 2, or complementsthereof and does not hybridize under stringent hybridization conditionswith a DNA molecule not comprising SEQ ID NO: 1, SEQ ID NO: 2, orcomplements thereof.
 11. A method of detecting the presence of a DNAmolecule derived from event KK179-2 in a DNA sample, the methodcomprising: a. contacting said DNA sample with the polynucleotide probeof claim 6; b. subjecting said DNA sample and said polynucleotide probeto stringent hybridization conditions; and c. detecting hybridization ofsaid polynucleotide probe to said DNA molecule derived from eventKK179-2 in said DNA sample.
 12. A pair of DNA molecules consisting of afirst DNA molecule and a second DNA molecule different from the firstDNA molecule, wherein said first and second DNA molecules comprise apolynucleotide molecule having a nucleotide sequence of sufficientlength of consecutive nucleotides of SEQ ID NO: 6, or a complementthereof, to function as DNA primers when used together in anamplification reaction with a template derived from event KK179-2 toproduce an amplicon diagnostic for event KK179-2 DNA in a sample.
 13. ADNA detection kit comprising at least one polynucleotide molecule ofsufficient length of consecutive nucleotides of SEQ ID NO: 6, orcomplements thereof, to function as a DNA primer or polynucleotide probespecific for detecting the presence of DNA derived from event KK179-2,wherein detection of said DNA is diagnostic for the presence of saidKK179-2 DNA in a sample.
 14. The DNA detection kit of claim 13, whereinat least one polynucleotide molecule is selected from the groupconsisting of SEQ ID NO: 1, and SEQ ID NO:
 2. 15. A microorganismcomprising the DNA molecule of claim
 7. 16. The microorganism of claim15, wherein said microorganism is a plant cell.